|Publication number||US5578302 A|
|Application number||US 08/430,297|
|Publication date||Nov 26, 1996|
|Filing date||Apr 28, 1995|
|Priority date||Jul 6, 1992|
|Also published as||CA2099855A1, CA2099855C, DE69223615D1, DE69223615T2, EP0577903A1, EP0577903B1|
|Publication number||08430297, 430297, US 5578302 A, US 5578302A, US-A-5578302, US5578302 A, US5578302A|
|Inventors||Dominique Brassart, Pierre Michetti, Jean-Richard Neeser|
|Original Assignee||Nestec S.A.|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (17), Non-Patent Citations (38), Referenced by (37), Classifications (22), Legal Events (3)|
|External Links: USPTO, USPTO Assignment, Espacenet|
This application is a continuation application of application Ser. No. 08/084,528, filed Jun. 29, 1993, now abandoned.
This invention relates to an antigastritis and/or antiulcer agent and to a lactic acid bacterium strain capable of producing this agent.
It is known that certain lactic acid bacterium strains show good adhesion to intestinal cells and, by virtue of this property, lend themselves to therapeutic applications.
European Patent Application Publication No. 199,535 (Gorbach and Goldin), for example, proposes a Lactobacillus strain named GG after its inventors and deposited in the ATCC (American Type Culture Collection) under No. 53103 which is intended to be administered to human beings or to animals for therapeutic or prophylactic treatment of the digestive tract.
The problem addressed by the present invention was to provide an antigastritis and/or antiulcer agent and a lactic acid bacterium strain capable of producing this agent which could be administered to human beings or to animals for therapeutic or prophylactic treatment of the stomach and particularly the pylorus.
To this end, the present invention relates to an antigastritis and/or antiulcer agent which is capable of displacing pathogenic bacteria from intestinal and/or gastric cells, to a biologically pure culture of a lactic acid bacterium strain selected for its ability to produce this agent and to a composition containing an effective quantity of the agent or the culture mentioned and an ingestible support, more particularly a pharmaceutically acceptable support and/or a food product, such as an acidified milk, more particularly a yogurt or a milk-based powder formulation.
It has been found that certain strains of lactic acid bacteria particularly strains of Lactobacillus acidophilus, are capable of displacing pathogenic bacteria, such as Heliobacter (H.) pylori for example, from the intestinal cells to which they adhere. It has also been found that the strains in question have the ability to produce an agent showing such power of displacement and, above all, to produce it in their culture medium.
Accordingly, the agent, the strain and the composition according to the invention are more particularly intended for administration to human beings or animals for therapeutic or prophylactic treatment of the stomach and particularly for the treatment of gastritis or ulcers of the stomach or the pylorum.
Among the various strains selected in accordance with the present invention, one was deposited by way of example under the Budapest Treaty on the Jun. 30, 1992 at the Collection Nationale de Microorganismes (CNCM), Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France, where it was given the No. Lactyobacillus acidophilus strain CNCM I-1225.
Subsequent to deposit of strain CNCM I-1225, the taxonomic classification of Lactobacillus acidophilus was reorganized to include six subgroups or "genomospecies." See Fujisawa, et al., Taxonomic Study of the Lactobacillus acidophilus Group with Recognition of Lactobacillus gallinarum sp nov and Lactobacillus johnsonii sp nov and Synonymy of Lactobacillus acidophilus Group A3 with the Type Strain of Lactobacillus amylovorous, Int. J. Syst. bacteriol. 42:487-491 (1992). Subsequent to that taxonomic reclassification, it was determined that strain CNCM I-1225 is a member of the newly established Lactobacillus johnsonii species.
Details of the morphology and general properties of this strain are given in the following:
Gram-positive microorganism, non-motile, non-sporing.
Fairly short and thick rodlets.
Microaerophilic microorganism with homofermentative metabolism giving rise to the production of L(+) and D(-) lactic acid.
Other characteristics: catalase (-), production of CO2 (-), hydrolysis of arginine (-).
Fermentation of sugars
Amygdaline (+), arabinose (-), cellobiose (+), esculine (+), fructose (+), galactose (+), glucose (+), lactose (+), maltose (+/-), mannitol (-), mannose (+), melibiose (-), raffinose (+), ribose (-), salicine (+), sucrose (+), trehalose (+).
Details of the particular properties for which the present strain may be selected are given in the following:
Adhesion to gastric cells may be compared with adhesion to intestinal cells providing the receptors recognized by a microorganism on the two types of cells are similar and the microorganism shows strong power of adhesion to the two types of cells.
Certain pathogenic microorganisms, such as Heliobacter pylori for example, seem to have this property.
It may be verified in particular that Helicobacter pylori is capable of adhering to human intestinal cells derived from adenocarcinomas, for example the HT29 cells (Pinto et al., Biol. Cell. 55, 193-196, 1982), in a monolayer culture in vitro.
To this end, the HT29 cells are cultured at 37° C. in Dulbeccos modified Eagle medium (DMEM) containing 10% galactose and dialyzed calf serum in an atmosphere of 10% CO2 and 90% air and are used before the 20th culture passage. The cultures are performed on degreased glass slips placed in 24-cup trays.
H. pylori is cultured on Muller-Hinton plates, 10% sheep's blood, for 72 h at 37° C. in an atmosphere of 5% O2, 10% CO2 and 85% nitrogen. The plates are scraped and the bacteria are collected and then washed in physiological solution.
H. pylori is inoculated onto the HT29 cells in a quantity of 106 viable germs or cells (cfu) per cm2. The whole is then incubated for 2 h at 37° C. and the monolayers are washed three times.
The number of H. pylori cells adhering to the HT29 cells is determined by a urease test (Jatrox-Hp-Test) of which the principle is that an aqueous solution of urea and phenol red changes color from yellow to fuschia pink in the presence of urease which catabolizes the production of basic metabolites of urea, ammonium and bicarbonate. The intensity of the reaction is read off from a spectrophotometer at 550 nm. This test is linear for values of 104 to 106 cfu/ml.
To determine the ability of a strain, culture, agent or composition according to the invention to displace pathogenic bacteria, the extent to which it is capable of displacing the H. pylori adhering to the HT29 cells, for example, may be examined.
To this end, a solution or suspension of the lactic acid bacterium to be selected or agent to be tested may be added to the HT29 cells to which the H. pylori cells adhere, after which the whole is incubated for 1 h and washed several times before the urease test is performed.
If a culture of the L. johnsonii strain CNCM I-1225, for example, is added with its culture medium (MRS or milk for example), 50% diluted with DMEM, and is added in a quantity of 107 cfu per cm2, it is found that, of the 2×106 cfu/cm2 of adhering H. pylori in the absence of competitive bacteria, only 6×103 cfu/cm2 remain after incubation for 1 h with the L. johnsonii strain CNCM I-1225.
This demonstrates the advantage which such a strain can have in the treatment of gastritis or ulcers of the stomach or pylorum.
Similarly, if the supernatant phase of the above L. johnsonii culture is added to the HT29 cells to which the H. pylori cells adhere, a very pronounced displacement of H. pylori is again observed. This shows that certain strains of lactic acid bacteria, in the present case the L. johnsonii strain CNCM I-1225, are capable of secreting an antigastritis and/or antiulcer agent in their culture medium. Accordingly, the supernatant phase and the agent extracted therefrom may themselves be of considerable advantage in the treatment of gastritis or ulcers of the stomach or the pylorum.
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|U.S. Classification||424/93.45, 435/854, 435/252.9, 435/853, 426/61|
|International Classification||C12R1/23, C12N1/20, A23C9/12, A61P1/00, A61P1/04, A23L1/00, C12P1/04, A23C9/123, A61K35/74|
|Cooperative Classification||Y10S435/854, Y10S435/853, A61K35/747, A23C9/1234, C12P1/04|
|European Classification||C12P1/04, A61K35/74A2B, A23C9/123D|
|May 15, 2000||FPAY||Fee payment|
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|May 16, 2008||FPAY||Fee payment|
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